Cystic Fibrosis Transmembrane Conductance Regulator Protein Research Articles

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Ubiquitylation as a Novel Pharmaceutical Target for Cystic Fibrosis.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene decrease the structural stability and function of the CFTR protein, resulting in cystic fibrosis. Recently, the effect of CFTR-targeting combination therapy has dramatically increased, and it is expected that add-on drugs that modulate the CFTR surrounding environment will further enhance their effectiveness. Various interacting proteins have been implicated in the structural stability of CFTR and, among them, molecules involved in CFTR ubiquitylation are promising therapeutic targets as regulators of CFTR degradation. This review focuses on the ubiquitylation mechanism that contributes to the stability of mutant CFTR at the endoplasmic reticulum (ER) and post-ER compartments and discusses the possibility as a pharmacological target for cystic fibrosis (CF).

A Peptide Nucleic Acid (PNA) Masking the miR-145-5p Binding Site of the 3'UTR of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) mRNA Enhances CFTR Expression in Calu-3 Cells.

Peptide nucleic acids (PNAs) have been demonstrated to be very useful tools for gene regulation at different levels and with different mechanisms of action. In the last few years the use of PNAs for targeting microRNAs (anti-miRNA PNAs) has provided impressive advancements. In particular, targeting of microRNAs involved in the repression of the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which is defective in cystic fibrosis (CF), is a key step in the development of new types of treatment protocols. In addition to the anti-miRNA therapeutic strategy, inhibition of miRNA functions can be reached by masking the miRNA binding sites present within the 3′UTR region of the target mRNAs. The objective of this study was to design a PNA masking the binding site of the microRNA miR-145-5p present within the 3′UTR of the CFTR mRNA and to determine its activity in inhibiting miR-145-5p function, with particular focus on the expression of both CFTR mRNA and CFTR protein in Calu-3 cells. The results obtained support the concept that the PNA masking the miR-145-5p binding site of the CFTR mRNA is able to interfere with miR-145-5p biological functions, leading to both an increase of CFTR mRNA and CFTR protein content.

Re-imagining cystic fibrosis care: next generation thinking.

Cystic fibrosis (CF) is a common multi-system genetically inherited condition, predominately found in individuals of Caucasian decent. Since the identification of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene in 1989, and the subsequent improvement in understanding of CF pathophysiology, significant increases in life-expectancy have followed. Initially this was related to improvements in the management and systems of care for treating the various affected organ systems. These cornerstone treatments are still essential for CF patients born today. However, over the last decade, the major advance has been in therapies that target the resultant genetic defect: the dysfunctional CFTR protein. Small molecule agents that target this dysfunctional protein via a variety of mechanisms have led to lung function improvements, reductions in pulmonary exacerbation rates and increases in weight and quality-of-life indices. As more patients receive these agents earlier and earlier in life, it is likely that general CF care will increasingly pivot around these specific therapies, although it is also likely that effects other than those identified in the initial trials will be discovered and need to be managed. Despite great excitement for modulator therapies, they are unlikely to be suitable or available for all; whether this is due to a lack of availability for specific CFTR mutations, drug-reactions or the health economic set-up in certain countries. Nevertheless, the CF community must be applauded for its ongoing focus on research and development for this life-limiting disease. With time, personalised individualised therapy would ideally be the mainstay of CF care.

42 CFTR Mediates Enteric Bacterial Translocation Post Severe Burn via ERK and Inflammatory Signaling

Abstract Introduction This research was to investigate the role of cystic fibrosis transmembrane conductance regulator (CFTR) in mediating enteric bacterial translocation (EBT) post severe burn as well as the signaling pathways involved. Methods A hypoxia treatment model of Caco-2 in vitro and a 30% TBSA full-thickness dermal burn mice model in vivo were established to explore the effect of major burn injury on intestinal epithelial barrier and the expression of CFTR and tight junction proteins. In addition, the DF508 mice (mice with F508del CFTR gene mutation) were used to analyze bacterial counts in extraintestinal organs and the expression of ZO-1, OCLN and E-cadherin. And CFTR specific inhibitor or down-regulation of CFTR in Caco-2 cells were used to explore the effect of CFTR on tight junction of intestinal cells. Furthermore, in the models above, MAPK pathway and inflammatory factors were detected to explore the mechanism. Finally, Vitamin D3 was used to pretreat mice before severe burn or Caco-2 cells before hypoxia. Bacterial counts in extraintestinal organs and the expression of CFTR, ZO-1, OCLN and P-ERK as well as the concentrations of inflammatory factors were detected. Results Our data showed a decrease of CFTR, concomitant with activation of ERK signaling, elevation of inflammatory factors (TNF-α, IL-1β and IL-8) and declined expression of ZO-1, occludin and E-cadherin, and a cellular mislocation of ZO-1 and OCLN post hypoxia. Furthermore, either administration of CFTR specific inhibitor or down-regulation of CFTR caused similar alterations as hypoxia did. Ileum of DF508 -/- and +/- mice exhibited increased inflammation, declined ZO-1, occludin and E-cadherin and EBT compared to +/+ mice. Moreover, vitamin D3 could reverse the junctional damage induced by hypoxia, thus preventing occurrence of EBT, possibly via up-regulating CFTR. Conclusions CFTR/ERK/inflammatory factors signaling was critical in modulating intestinal epithelial junction, accordingly regulating EBT. Vitamin D3 can protect intestinal epithelial barrier after severe burn, which may be associated with the up-regulation of CFTR, the inhibition of ERK pathway and decreased pro-inflammatory cytokine. Applicability of Research to Practice This research may be used in practice in the future to regulate the metabolism after severe burns.

CFTR Modulators: The Changing Face of Cystic Fibrosis in the Era of Precision Medicine.

Cystic fibrosis (CF) is a lethal inherited disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which result in impairment of CFTR mRNA and protein expression, function, stability or a combination of these. Although CF leads to multifaceted clinical manifestations, the respiratory disorder represents the major cause of morbidity and mortality of these patients. The life expectancy of CF patients has substantially lengthened due to early diagnosis and improvements in symptomatic therapeutic regimens. Quality of life remains nevertheless limited, as these individuals are subjected to considerable clinical, psychosocial and economic burdens. Since the discovery of the CFTR gene in 1989, tremendous efforts have been made to develop therapies acting more upstream on the pathogenesis cascade, thereby overcoming the underlying dysfunctions caused by CFTR mutations. In this line, the advances in cell-based high-throughput screenings have been facilitating the fast-tracking of CFTR modulators. These modulator drugs have the ability to enhance or even restore the functional expression of specific CF-causing mutations, and they have been classified into five main groups depending on their effects on CFTR mutations: potentiators, correctors, stabilizers, read-through agents, and amplifiers. To date, four CFTR modulators have reached the market, and these pharmaceutical therapies are transforming patients' lives with short- and long-term improvements in clinical outcomes. Such breakthroughs have paved the way for the development of novel CFTR modulators, which are currently under experimental and clinical investigations. Furthermore, recent insights into the CFTR structure will be useful for the rational design of next-generation modulator drugs. This review aims to provide a summary of recent developments in CFTR-directed therapeutics. Barriers and future directions are also discussed in order to optimize treatment adherence, identify feasible and sustainable solutions for equitable access to these therapies, and continue to expand the pipeline of novel modulators that may result in effective precision medicine for all individuals with CF.

Phenotypic Characterization and Comparison of Cystic Fibrosis Rat Models Generated Using CRISPR/Cas9 Gene Editing

Animal models of cystic fibrosis (CF) are essential for investigating disease mechanisms and trialing potential therapeutics. This study generated two CF rat models using clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 gene editing. One rat model carries the common human Phe508del (ΔF508) CF transmembrane conductance regulator (CFTR) mutation, whereas the second is a CFTR knockout model. Phenotype was characterized using a range of functional and histologic assessments, including nasal potential difference to measure electrophysiological function in the upper airways, RNAscope in situ hybridization and quantitative PCR to assess CFTR mRNA expression in the lungs, immunohistochemistry to localize CFTR protein in the airways, and histopathologic assessments in a range of tissues. Both rat models revealed a range of CF manifestations, including reduced survival, intestinal obstruction, bioelectric defects in the nasal epithelium, histopathologic changes in the trachea, large intestine, and pancreas, and abnormalities in the development of the male reproductive tract. The CF rat models presented herein will prove useful for longitudinal assessments of pathophysiology and therapeutics.

Amplifiers co-translationally enhance CFTR biosynthesis via PCBP1-mediated regulation of CFTR mRNA

BackgroundCystic fibrosis (CF) is a recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We previously described a first-in-class CFTR modulator that functions as an amplifier to selectively increase CFTR expression and function. The amplifier mechanism is distinct from and complementary to corrector and potentiator classes of CFTR modulators. Here we characterize the mechanism by which amplifiers increase CFTR mRNA, protein, and activity. MethodsBiochemical studies elucidated the action of amplifiers on CFTR mRNA abundance and translation and defined the role of an amplifier-binding protein that was identified using chemical proteomics. ResultsAmplifiers stabilize CFTR mRNA through a process that requires only the translated sequence of CFTR and involves translational elongation. Amplifiers enrich ER-associated CFTR mRNA and increase its translational efficiency through increasing the fraction of CFTR mRNA associated with polysomes. Pulldowns identified the poly(rC)-binding protein 1 (PCBP1) as directly binding to amplifier. A PCBP1 consensus element was identified within the CFTR open reading frame that binds PCBP1. This sequence proved necessary for amplifier responsiveness. ConclusionsSmall molecule amplifiers co-translationally increase CFTR mRNA stability. They enhance translation through addressing the inherently inefficient membrane targeting of CFTR mRNA. Amplifiers bind directly to PCBP1, show enhanced affinity in the presence of bound RNA, and require a PCBP1 consensus element within CFTR mRNA to elicit translational effects. These modulators represent a promising new and mechanistically novel class of CFTR therapeutic. They may be useful as a monotherapy or in combination with other CFTR modulators.

Dynamic Regulation of Bicarbonate Permeability through CFTR Channel by WNK1

Increase in relative HCO3− permeability (PHCO3/PCl) of cystic fibrosis transmembrane conductance regulator (CFTR) channel by[Cl−]i-sensitive kinases is essential for pancreatic HCO3− secretion. Defects in pancreatic HCO3− secretion caused by CFTR mutations are associated with cystic fibrosis and pancreatitis. However, the molecular mechanisms by which [Cl−]i-sensitive kinases regulate CFTR anion selectivity are unknown. We examined the mechanisms of CFTR PHCO3/PCl regulation by [Cl−]i-sensitive kinases using the electrophysiological and molecular approach, including whole-cell, outside-out, and inside-out patch clamp recordings and molecular dissection of WNK1 and CFTR proteins. Moreover, we analyzed the effects of pancreatitis-causing CFTR mutations on the WNK1-mediated regulation of CFTR. Although SPAK and/or OSR, which constitute a [Cl−]i-sensitive kinase cascade, wasn't expressed, the expression of WNK1 alone was sufficient to increase the CFTR PHCO3/PCl in patch clamp recordings. In molecular dissection of the WNK1 protein, the WNK1 kinase domain contributes CFTR PHCO3/PCl regulation by direct association with CFTR, while the surrounding N-terminal regions regulate the [Cl−]i-sensitivity of WNK1. Therefore, the inclusion of WNK1 N-terminal recombinant proteins in excised patch clamp recordings increased the CFTR PHCO3/PCl in a dose-dependent manner. The pancreatitis-causing R74Q and R75Q mutations in elbow helix 1 of CFTR hampered WNK1-CFTR physical associations and reduced WNK1-mediated CFTR PHCO3/PCl regulation. The CFTR PHCO3/PCl is regulated by [Cl−]i and a WNK1-dependent mechanism. In low [Cl−]i, physical associations between the N-terminal region of WNK1 and CFTR near p.R75 increase PHCO3/PCl. Defects in this process underlie the pathogenesis of some CFTR-related disorders.

Genetic diagnosis in practice: From cystic fibrosis to CFTR-related disorders

Cystic fibrosis (CF) is a channelopathy caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) protein. Diagnosis of CF has long relied on a combination of clinical (including gastrointestinal and/or respiratory) symptoms and elevated sweat chloride concentration. After cloning of the CFTR gene in 1989, genetic analysis progressively became an important aspect of diagnosis. Although combination of sweat test and genetic analysis have simplified the diagnosis of CF in most cases, difficult situations remain, especially in cases that do not fulfill all diagnostic criteria. Such situations are most frequently encountered in patients presenting with a single-organ disease (e.g., congenital absence of the vas deferens, pancreatitis, bronchiectasis) leading to a diagnosis of CFTR-related disorder, or when the presence/ absence of CF is not resolved after newborn screening. This article reviews the diagnostic criteria of CF, with special emphasis on genetic testing. © 2020 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.

Gating Dynamics, Regulation and Pharmacology of the CFTR Anion Channel

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) belongs into the C subfamily of ABC proteins, but its transmembrane domains (TMDs) form an anion selective channel pore which is under the control of its three cytosolic domains. Phosphorylation by protein kinase A (PKA) of multiple serines in the unique regulatory (R) domain is a prerequisite for channel activity, and gating of phosphorylated channels is driven by ATP binding and hydrolysis at the two cytosolic nucleotide binding domains (NBDs). Anion transport through CFTR is indispensable for the salt-water balance of epithelia lining the lung, gut, pancreatic duct, and sweat duct, and mutations that impair CFTR surface expression, gating, or anion permeation through the open pore cause cystic fibrosis (CF). In the past decade small molecule potentiator drugs, that enhance the open probability of mutant CFTR channels, have found their way to the clinics, and high-resolution structures of the CFTR protein in various conformations, obtained by electron-cryomicroscopy, have shed unprecedented light on CFTR molecular mechanisms. This talk will focus on recent advances in our understanding of three aspects of CFTR function: the dynamics of the pore opening transition in phosphorylated ATP-bound channels, the kinetic mechanism of the potentiator drug Vx-770 (ivacaftor), and the molecular mechanism of physiological gating regulation through interaction of CFTR with the catalytic subunit of PKA.

A SYK/SHC1 pathway regulates the amount of CFTR in the plasma membrane.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the recessive genetic disease cystic fibrosis, where the chloride transport across the apical membrane of epithelial cells mediated by the CFTR protein is impaired. CFTR protein trafficking to the plasma membrane (PM) is the result of a complex interplay between the secretory and membrane recycling pathways that control the number of channels present at the membrane. In addition, the ion transport activity of CFTR at the PM is modulated through post-translational protein modifications. Previously we described that spleen tyrosine kinase (SYK) phosphorylates a specific tyrosine residue in the nucleotide-binding domain 1 domain and this modification can regulate the PM abundance of CFTR. Here we identified the underlying biochemical mechanism using peptide pull-down assays followed by mass spectrometry. We identified in bronchial epithelial cells that the adaptor protein SHC1 recognizes tyrosine-phosphorylated CFTR through its phosphotyrosine-binding domain and that the formation of a complex between SHC1 and CFTR is induced at the PM in the presence of activated SYK. The depletion of endogenous SHC1 expression was sufficient to promote an increase in CFTR at the PM of these cells. The results identify a SYK/SHC1 pathway that regulates the PM levels of CFTR channels, contributing to a better understanding of how CFTR-mediated chloride secretion is regulated.

Potentially lethal cystic fibrosis gene variant in the orangutan.

A syndrome of chronic upper and lower airway disease leading to increased morbidity and mortality occurs primarily in captive orangutans. Similarities in symptoms to the inherited human respiratory disease, cystic fibrosis, led us to hypothesize that orangutan respiratory disease is a result of variants in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene. We identified the nonsense variant, c.484A>T (p.Lys162X), in heterozygosity in an unaffected orangutan. Analysis of the pedigree of this orangutan confirmed that both his sire and deceased fetus also harbored the c.484A>T allele. An expression minigene harboring c.484A>T produced no full-length CFTR protein in HEK293 cells. Finally, the c.484A>T CFTR messenger RNA abundance was severely reduced in primary nasal epithelial cells of the orangutan indicating that c.484A>T (p.Lys162X) is potentially lethal. Genetic screening of the captive orangutan population could be used to prevent transmission of this potentially lethal variant, and thus aid in the conservation of this critically endangered species.

Correcting airways in cystic fibrosis

Gene Therapy Cystic fibrosis is caused by inactivating mutations in the cystic fibrosis transmembrane conductance regulator ( CFTR ) gene. Deletion of phenylalanine-508 is the most common mutation. Vaidyanathan et al. used CRISPR-associated protein 9 (Cas9)–mediated CFTR editing and adeno-associated virus delivery to correct the phenylalanine-508 deletion in upper-airway basal stem cells from 10 patients with cystic fibrosis. They achieved 30 to 50% gene correction and improved CFTR protein function. The corrected cells successfully engrafted within a clinically approved scaffold. This finding offers possibilities for clinical development of upper airway implants to treat respiratory failure, which is the biggest cause of mortality in cystic fibrosis patients. Cell Stem Cell 10.1016/j.stem.2019.11.002 (2019).

The pathological features of cystic fibrosis and the diagnostic techniques and treatments involved

Cystic fibrosis is a disease found predominantly in Caucasians. It is caused by an autosomal recessive mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The mutation(s) lead to defective CFTR proteins, or transmembrane cyclic adenosine monophosphate (cAMP)-dependent chloride channels found on the surface of epithelial cells. The respiratory system is most impacted by this abnormal phenotype, and lung disease can bring about the majority of the various issues that a cystic fibrosis patient has to deal with. Even so, due to the excess amount of mucus that is secreted, individuals diagnosed with cystic fibrosis experience complications in other organs as well. Today, newborn screening is widely used as a means of early diagnosis of cystic fibrosis, and there are also multiple other novel technologies that aim for accurate diagnosis and prevention of the disease. In addition, while traditional treatment programs are based on curing the symptoms that the patients exhibit, new regimens are directed at correcting the error that occurred at the molecular level. With improvements made in the medical field, cystic fibrosis patients are able to live longer and have a better quality of life. This review article serves as a compilation of the current knowledge on the causes of cystic fibrosis, the physiological processes associated with the disease, the techniques for diagnosis of the disease, and finally the recent and upcoming therapeutics for the disease.

Promoter hypermethylation of the CFTR gene as a novel diagnostic and prognostic marker of breast cancer.

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-related deaths among women. New biomarkers with definite diagnostic and prognostic efficacy are urgently needed. Here, we showed that the promoter of the cystic fibrosis transmembrane conductance regulator (CFTR) was hypermethylated in breast cancer. The messenger RNA level of CFTR was downregulated in breast cancer. Notably, all 19 breast cancer patients with hypermethylated CFTR were diagnosed with invasive carcinoma. Moreover, CFTR was upregulated in decitabine (10 μM) treated breast cancer cells. Overexpression of CFTR inhibited cell growth whereas knockdown of CFTR promoted cell invasion. In the tissue array analysis, the CFTR protein level decreased significantly in breast cancer and low CFTR protein level correlated with poor survival with a P-value of 0.034. Thus, promoter hypermethylation of the CFTR gene might be a novel diagnostic marker of breast cancer.

132 Introduction of F508del human mutation into the CFTR gene of sheep fetal fibroblasts using CRISPR/Cas9 ribonucleoprotein

Cystic fibrosis (CF) is an autosomal recessive genetic disease that affects over 30 000 people in the United States and is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR protein is a cAMP-regulated C− channel responsible for regulation of anion transport, primarily in the epithelial cells. We have previously generated a sheep model of CF by genetically inactivating the CFTR gene (Fan et al. 2018 JCI Insight 3, e123529). The newborn CFTR −/− sheep develops severe disease consistent with CF pathology in humans. The CF model is extremely valuable for understanding the developmental aspects of CF disease, as sheep have been used extensively in the study of human fetal growth and development. Sheep, like humans, typically give birth to only one or two offspring in each pregnancy, which make them more suitable than many other species for testing prenatal gene-editing treatments. Thus, in this new study, we are working on the generation of F508del sheep CF model. The F508del mutation was chosen because it is the most common mutation in the human CFTR gene (~70%). This mutation is characterised by the deletion of the CTT nucleotides, which ultimately deletes the phenylalanine residue at position 508. The F508del mutation causes misfolding of the CFTR protein, which is further degraded by proteases. Even though several CFTR modulators are available, they are not effective in all patients. Additionally, they cannot reverse deleterious prenatal CF manifestations. Hence, this model will be valuable for evaluating both prenatal drug and gene therapies. Here, we used a CRISPR/Cas9 gene-editing approach to introduce the F508del mutation into the sheep genome. We designed an sgRNA targeting exon 11 of the sheep CFTR gene using the Benchling software (https://benchling.com/academic). The sgRNA was synthesised by Synthego and Cas9 purchased from ThermoFisher. Using the Lonza-4D-Nucleofector system, Cas9/sgRNA ribonucleoprotein complex was transfected into sheep fetal fibroblasts (SFFs), along with 100bp single-stranded oligodeoxynucleotide, flanking the F508del mutation, for the homology-directed repair. The transfected cells were subsequently cultured in Dulbecco's modified Eagle's medium, supplemented with 15% fetal bovine serum and 1% penicillin, and incubated at 38.5°C. Two days post-transfection, SFFs were seeded individually into five 96-well plates by limited dilution. After seven days, the individual colonies were expanded into 24-well plates and cultured for three more days. A total of 56 single-cell-derived SFF colonies were isolated. The presence of F508del mutation was confirmed by amplifying the PCR products of the exon 11 flanking the mutation site and subjecting each amplicon to Sanger sequencing. The sequencing results indicated that the indels (insertion/deletion) were introduced in 49 out of 56 (87.5%) of the colonies, and four (7.14%) of them were confirmed to have biallelic F508del mutations based on sequencing peaks. Therefore, we successfully introduced the F508del mutation in SFFs that will be used for the production of F508del CF sheep by somatic cell nuclear transfer.

In Situ Analysis Reveals That CFTR Is Expressed in Only a Small Minority of β-Cells in Normal Adult Human Pancreas.

Although diabetes affects 40% to 50% of adults with cystic fibrosis, remarkably little is known regarding the underlying mechanisms leading to impaired pancreatic β-cell insulin secretion. Efforts toward improving the functional β-cell deficit in cystic fibrosis-related diabetes (CFRD) have been hampered by an incomplete understanding of whether β-cell function is intrinsically regulated by cystic fibrosis transmembrane conductance regulator (CFTR). Definitively excluding meaningful CFTR expression in human β-cells in situ would contribute significantly to the understanding of CFRD pathogenesis. To determine CFTR messenger ribonucleic acid (mRNA) and protein expression within β-cells in situ in the unmanipulated human pancreas of donors without any known pancreatic pathology. In situ hybridization for CFTR mRNA expression in parallel with insulin immunohistochemical staining and immunofluorescence co-localization of CFTR with insulin and the ductal marker, Keratin-7 (KRT7), were undertaken in pancreatic tissue blocks from 10 normal adult, nonobese deceased organ donors over a wide age range (23-71 years) with quantitative image analysis. CFTR mRNA was detectable in a mean 0.45% (range 0.17%-0.83%) of insulin-positive cells. CFTR protein expression was co-localized with KRT7. One hundred percent of insulin-positive cells were immunonegative for CFTR. For the first time, in situ CFTR mRNA expression in the unmanipulated pancreas has been shown to be present in only a very small minority (<1%) of normal adult β-cells. These data signal a need to move away from studying endocrine-intrinsic mechanisms and focus on elucidation of exocrine-endocrine interactions in human cystic fibrosis.

MiR-636: A Newly-Identified Actor for the Regulation of Pulmonary Inflammation in Cystic Fibrosis.

Cystic fibrosis (CF) results from deficient CF transmembrane conductance regulator (CFTR) protein activity leading to defective epithelial ion transport. Pulmonary degradation due to excessive inflammation is the main cause of morbidity and mortality in CF patients. By analysing miRNAs (small RNAseq) in human primary air-liquid interface cell cultures, we measured the overexpression of miR-636 in CF patients compared to non-CF controls. We validated these results in explant biopsies and determined that the mechanism underlying miR-636 overexpression is linked to inflammation. To identify specific targets, we used bioinformatics analysis to predict whether miR-636 targets the 3′-UTR mRNA regions of IL1R1 and RANK (two pro-inflammatory cytokine receptors), IKBKB (a major protein in the NF-κB pathway), and FAM13A (a modifier gene of CF lung phenotype implicated in epithelial remodelling). Using bronchial epithelial cells from CF patients to conduct a functional analysis, we showed a direct interaction between miR-636 and IL1R1, RANK, and IKBKB, but not with FAM13A. These interactions led to a decrease in IL1R1 and IKKβ protein expression levels, while we observed an increase in RANK protein expression levels following the overexpression of miR-636. Moreover, NF-κB activity and IL-8 and IL-6 secretions decreased following the transfection of miR-636 mimics in CF cells. Similar but opposite effects were found after transfection with an antagomiR-636 in the same cells. Furthermore, we demonstrated that miR-636 was not regulated by Pseudomonas aeruginosa in our model. We went on to show that miR-636 is raised in the blood neutrophils, but not in the plasma, of CF patients and may have potential as a novel biomarker. Collectively, our findings reveal a novel actor for the regulation of inflammation in CF, miR-636, which is able to reduce constitutive NF-κB pathway activation when it is overexpressed.

Biological Characterization of F508delCFTR Protein Processing by the CFTR Corrector ABBV-2222/GLPG2222.

Cystic fibrosis (CF) is the most common monogenic autosomal recessive disease in Caucasians caused by pathogenic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (CFTR). Significant small molecule therapeutic advances over the past two decades have been made to target the defective CFTR protein and enhance its function. To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in CF, two biomolecular activities are required, namely, correctors to increase the amount of properly folded F508delCFTR levels at the cell surface and potentiators to allow the effective opening, i.e., function of the F508delCFTR channel. Combined, these activities enhance chloride ion transport yielding improved hydration of the lung surface and subsequent restoration of mucociliary clearance. To enhance clinical benefits to CF patients, a complementary triple combination therapy consisting of two corrector molecules, type 1 (C1) and type 2, with additive mechanisms along with a potentiator are being investigated in the clinic for maximum restoration of mutated CFTR function. We report the identification and in vitro biologic characterization of ABBV-2222/GLPG2222 (4-[(2R,4R)-4-({[1-(2,2-difluoro-1,3-benzodioxol-5-yl)cyclopropyl]carbonyl}amino)-7-(difluoromethoxy)-3,4-dihydro-2H-chromen-2-yl]benzoic acid),-a novel, potent, and orally bioavailable C1 corrector developed by AbbVie-Galapagos and currently in clinical trials-which exhibits substantial improvements over the existing C1 correctors. This includes improvements in potency and drug-drug interaction (DDI) compared with 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (VX-809, Lumacaftor) and improvements in potency and efficacy compared with 1-(2,2-difluoro-1,3-benzodioxol-5-yl)-N-[1-[(2R)-2,3-dihydroxypropyl]-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)indol-5-yl]cyclopropane-1-carboxamide (VX-661, Tezacaftor). ABBV-2222/GLPG2222 exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR with an EC50 value <10 nM. SIGNIFICANCE STATEMENT: To address the most prevalent defect of the defective CFTR protein (i.e., F508del mutation) in cystic fibrosis, AbbVie-Galapagos has developed ABBV-2222/GLPG2222, a novel, potent, and orally bioavailable C1 corrector of this protein. ABBV-2222/GLPG2222, which is currently in clinical trials, exhibits potent in vitro functional activity in primary patient cells harboring F508del/F508del CFTR and substantial improvements over the existing C1 correctors.

An Intriguing Involvement of Mitochondria in Cystic Fibrosis.

Cystic fibrosis (CF) occurs when the cystic fibrosis transmembrane conductance regulator (CFTR) protein is not synthetized and folded correctly. The CFTR protein helps to maintain the balance of salt and water on many body surfaces, such as the lung surface. When the protein is not working correctly, chloride becomes trapped in cells, then water cannot hydrate the cellular surface and the mucus covering the cells becomes thick and sticky. Furthermore, a defective CFTR appears to produce a redox imbalance in epithelial cells and extracellular fluids and to cause an abnormal generation of reactive oxygen species: as a consequence, oxidative stress has been implicated as a causative factor in the aetiology of the process. Moreover, massive evidences show that defective CFTR gives rise to extracellular GSH level decrease and elevated glucose concentrations in airway surface liquid (ASL), thus encouraging lung infection by pathogens in the CF advancement. Recent research in progress aims to rediscover a possible role of mitochondria in CF. Here the latest new and recent studies on mitochondrial bioenergetics are collected. Surprisingly, they have enabled us to ascertain that mitochondria have a leading role in opposing the high ASL glucose level as well as oxidative stress in CF.